Transformation of seven species of filamentous fungi using the nitrate reductase gene of Aspergillus nidulans

Daboussi, Marie‐Josée; Djeballi, A.; Gerlinger, Catherine; Blaiseau, Pierre-Louis; Bouvier, Isabelle; Cassan, M; Lebrun, Marc; Parisot, Denise; Brygoo, Yves

doi:10.1007/bf00376803

Cited by 117 publications

(59 citation statements)

References 23 publications

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“…Media composition, M. grisea maintenance, transformation, and sexual crosses were as described (13,14). P1.2 (MAT1.2) and M4 (MAT1.1) are M. grisea strains from the Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD).…”

Section: Methodsmentioning

confidence: 99%

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PLS1 , a gene encoding a tetraspanin-like protein, is required for penetration of rice leaf by the fungal pathogen Magnaporthe grisea

Clergeot

Gourgues

Cots

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

154

152

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We describe in this study punchless, a nonpathogenic mutant from the rice blast fungus M. grisea, obtained by plasmid-mediated insertional mutagenesis. As do most fungal plant pathogens, M. grisea differentiates an infection structure specialized for host penetration called the appressorium. We show that punchless differentiates appressoria that fail to breach either the leaf epidermis or artificial membranes such as cellophane. Cytological analysis of punchless appressoria shows that they have a cellular structure, turgor, and glycogen content similar to those of wild type before penetration, but that they are unable to differentiate penetration pegs. The inactivated gene, PLS1, encodes a putative integral membrane protein of 225 aa (Pls1p). A functional Pls1p-green fluorescent protein fusion protein was detected only in appressoria and was localized in plasma membranes and vacuoles. Pls1p is structurally related to the tetraspanin family. In animals, these proteins are components of membrane signaling complexes controlling cell differentiation, motility, and adhesion. We conclude that PLS1 controls an appressorial function essential for the penetration of the fungus into host leaves. P lant pathogenic fungi are a major threat for crop production worldwide. Several of these fungal pathogens depend on the formation of a specialized cell, called an appressorium, for the successful penetration of host plant surfaces (1, 2). This penetration process is typical of fungal leaf spots such as rice blast caused by the filamentous ascomycete Magnaporthe grisea (3). This disease is disseminated by water-splashed conidia (ref. 4; Fig. 1A). After their adhesion to the host leaf by means of a strong glue (ref. 5; Fig. 1B), conidia germinate and differentiate appressoria at the tips of their germ tubes (Fig. 1C). This developmental process is induced by chemical and physical stimuli from the host surface such as hardness, hydrophobicity, or cutin monomers (2, 3). This dome-shaped, thick-walled, and melanized cell (Fig. 1D) promotes the differentiation of a specialized hypha called the penetration peg that is involved in penetration of plant surfaces (ref. 6; pp in Fig. 1E). The differentiation of appressoria and penetration pegs is the result of complex morphogenetic processes that involve major developmental and metabolic changes (2,3,6). Inside the epidermal plant cell, the penetration peg differentiates into a bulbous infection hypha that invades the whole cell and colonizes the leaf.To isolate genes involved in this infection process, we generated a collection of M. grisea pathogenicity mutants by using plasmid-mediated insertional mutagenesis. This strategy was successful for the cloning of pathogenicity genes in Colletotrichum lindemuthianum (7), Ustilago maydis,** and M. grisea (8,9). In this study, we isolated punchless, a nonpathogenic mutant defective in penetration of plant surfaces. We show that the PLS1 gene is inactivated in punchless and encodes a putative membrane protein related to the tetraspanin family (10...

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Section: Methodsmentioning

confidence: 99%

“…pBLHYG plasmid is a pBL plasmid with the hph cassette from pCB1003 (20) cloned at its EcoRI site. M. grisea genomic DNA extractions were performed as described (13). Total RNA was extracted by using the hot acid͞phenol protocol (21).…”

Section: Methodsmentioning

confidence: 99%

PLS1 , a gene encoding a tetraspanin-like protein, is required for penetration of rice leaf by the fungal pathogen Magnaporthe grisea

Clergeot

Gourgues

Cots

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

154

152

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“…Conversely, the niaD gene of A. nidulans, can complement NR-deficient strains of several other ascomycetous fungi (Daboussi et al 1989). The enzyme is strongly conserved through evolution and, as shown above, NR encoded by the P. infestans niaA gene contains all the conserved domains which are essential for enzyme activity.…”

Section: Comparison Of P Infestans Nr With That Of Other Organismsmentioning

confidence: 93%

“…This was done by transforming the P infestans niaA gene to a A. nidulans nia-mutant and analyzing the transformants for functional complementation of the NR deficiency. It has been shown that NR deficiency in several filamentous fungi, including some plant pathogens, can be complemented by the introduction of heterologous NR genes (Daboussi et al 1989;Whitehead et al 1989;Unkles et al 1989a,b). In a second approach to test oomycete regulatory sequences in a non-oomycete we used constructs containing promoter-GUS fusions.…”

Section: Introductionmentioning

confidence: 99%

NiaA, the structural nitrate reductase gene of Phytophthora infestans: isolation, characterization and expression analysis in Aspergillus nidulans

et al. 1995

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The nitrate reductase (NR) gene niaA of the oomycete Phytophthora infestans was selected from a gene library by heterologous hybridization. NiaA occurs as a single-copy gene and its expression is regulated by the nitrogen source. The nucleotide sequence of niaA was determined and comparison of the deduced amino-acid sequence of 902 residues with NRs of higher fungi and plants revealed a significant homology, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The P. infestans niaA gene was used as a model gene to test whether oomycete genes are functional in the ascomycete Aspergillus nidulans, a fungus which is highly accessible for molecular genetic studies. The complete niaA gene was stably integrated into the genome of a niadeletion mutant of A. nidulans. However, transformants containing one or more copies of the niaA gene were not able to complement the nia-mutant. This suggests that there is no functional expression of the introduced niaA gene in A. nidulans. In addition, the activity of two other oomycete gene promoters was analyzed in a transient expression assay. Plasmids containing chimaeric genes with the promoter of the P. infestans ubiquitin gene ubi3R, or the Bremia lactucae ham34 gene, fused to the coding sequence of the Escherichia coli P-glucuronidase (GUS) reporter gene, were transferred to A. nidulans protoplasts. No significant GUS activity was detectable indicating that the ubi3R and ham34 promoters are not active in A. nidulans. Apparently, the regulatory sequences which are sufficient for gene activation in oomycetes are not functional in the ascomycete A. nidulans.C. M. J. Pietersel J. van't Klooster G. C. M. van den Berg-Velthuis F. Govers (2i)

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“…It is characterized by its black spore, by which it is named after (Daboussi, Djeballi et al 1989). Aspergillus niger is used for the commercial production of both small molecules and proteins.…”

Section: Aspergillus Niger Expression Systemmentioning

confidence: 99%

Characterization of Recombinant Chloroperoxidase, and F103A and C29H/C79H/C87H Mutants

Zheng¹

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Transformation of seven species of filamentous fungi using the nitrate reductase gene of Aspergillus nidulans

Cited by 117 publications

References 23 publications

PLS1 , a gene encoding a tetraspanin-like protein, is required for penetration of rice leaf by the fungal pathogen Magnaporthe grisea

PLS1 , a gene encoding a tetraspanin-like protein, is required for penetration of rice leaf by the fungal pathogen Magnaporthe grisea

NiaA, the structural nitrate reductase gene of Phytophthora infestans: isolation, characterization and expression analysis in Aspergillus nidulans

Characterization of Recombinant Chloroperoxidase, and F103A and C29H/C79H/C87H Mutants

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